Do intracellular temperature gradients regulate cellular functions? It is fundamental to our understanding of biochemistry that enzymatic reactions are temperature-dependent, yet we tend to presume that intracellular temperature gradients, if they occur at all, must be dissipated so quickly as to be inconsequential. We hypothesize that intracellular temperature gradients may be as significant as ion gradients (e.g. Ca++ or pH) in regulating cellular functions. The challenge in addressing this question is to establish methodology for measuring meaningful changes in temperature at the subcellular level and to identify a biological process in which the role of this hypothesized temperature regulation can readily be assessed. We are developing methodology for measuring subcellular temperature changes in macrophages and applying these measurements to the process of phagocytosis, a process that strongly activates the cells and one in which degradative enzymes are enlisted to kill potentially threatening microbes. Preliminary data presented demonstrate that significant Phagosomal Heating occurs. The project is innovative in that intracellular temperature variations have not yet been implicated as important for regulating cellular functions. With this project, we intend to add a novel method of signaling to our understanding of molecular and cellular biology regulatory mechanisms. The approaches developed will be applicable to all areas molecular and cellular biology.